家蚕耐氟近等基因系差异表达基因的研究
|
摘要
我国是世界蚕业的发源地,栽桑养蚕、缫丝织绸已有5000多年的历史。栽桑养蚕是中国农业的一个重要组成部分,在国民经济中,已形成了一个农工贸完整的丝绸产业体系。蚕丝的出口贸易在我国国民经济中占有重要地位。蚕桑业的稳定发展对我国经济的发展具有深远的影响。
家蚕氟化物耐受性是很重要的一项经济性状。在蚕业生产中,家蚕是寡食性昆虫,以桑叶作为食物来源。桑叶受到氟化物的污染后造成家蚕大量死亡,对蚕桑业造成严重的危害。因此,研究家蚕的中毒机理,筛选研究家蚕耐氟基因,培育家蚕耐氟品种,并对耐氟主效基因进行定位克隆,十分重要。
本研究采用家蚕耐氟品种T6和氟化物敏感品种733新构建耐氟近等基因系。以构建好的近等基因系为材料,解剖获取回交11代的家蚕耐氟近等基因系群体中的耐氟个体和敏感个体中肠,构建家蚕耐氟近等基因系抑制消减杂交(SSH)cDNA文库。通过对抑制消减杂交cDNA文库中随机挑选的153个阳性克隆测序和聚类拼接,得到19个叠联群(contig)、48个已知功能基因(unigene)和28个未知功能基因。获得的表达序列标签(EST)经Blast比对和同源性分析,初步发现这些基因参与家蚕体内物质转运、能量代谢、基因转录、蛋白质合成与降解、蛋白质加工、信号转导、机体免疫防御、细胞结构形成等诸多生物学过程。采用实时定量RT-PCR方法,对部分基因在家蚕耐氟近等基因系群体中的耐氟个体和敏感个体的不同组织的表达进行定量分析。结果发现,磷酸根离子转运蛋白基因、ATP合成酶基因、液胞型H+-ATP酶基因以及主要过敏原蛋白基因在中肠、马氏管、脂肪体中的相对表达量有较明显的上调。实验结果可为进一步阐明家蚕耐氟的分子机制打下基础。
通过蛋白双向凝胶电泳及质谱技术,对家蚕耐氟近等基因系蛋白水平上的表达差异进行分析,最终得到了5个质谱鉴定结果可信的差异蛋白点,其中有2个蛋白点与家蚕的能量代谢和免疫防御有关,研究结果为今后展开蛋白功能方面的深入研究提供了一定基础。
China is the origin of sericulture in the world. The silk industry in our country have over 5,000 years of history, includeing many sections, such as planting mulberry, feeding silkworms, reeling silk from cocoons, knitting the silk and processing garment. Sericulture is a very important part of Chinese agriculture. Silk industry is a complete system composed of agriculture, industry and commerce. Silk exportation holds an important position in Chinese national economy.
The fluoride tolerance is the one of important economy traits in silkworm. Mulberry leaves are the major source of food for silkworm.It caused a large number of deaths to the silkworm when mulberry leaves were polluted by fluoride. This could cause serious harm to the sericulture. So it is very important to do works such as studying poisoning mechanism of fluoride in silkworm, searching for dominant endurance to fluoride(Def)gene of silkworm, breeding new new varieties of silkworm which are endurable to fluoride, map-based cloning of Def gene.
Silkworm near-isogenic lines(NILs)of dominant endurance to fluoride(Def)gene were constructed using silkworm strain 733 xin (sensitive to fluoride) and T6 (endurable to fluoride). An SSH (suppression subtractive hybridization) cDNA library was constructed between individuals which were sensitive to fluoride and individuals which were endurable to fluoride of BC11 progeny using PCR-selectTM cDNA Subtraction Kit. Plasmids were extracted from 153 positive clones randomly selected from the library and then sequenced and assembled using CAP3 software. 19contigs and 48 unigenes were obtained. Blastx searches and homology analysis with existing expressed sequence tags(ESTs)revealed that these genes were involved in many biological processes such as substance transportation, energy metabolism, gene transcription, protein synthesis or degradation, protein processing, signal transduction, organismal immunologic defence and formation of cellular construction etc. Real-time qPCR analysis confirmed that some genes, such as phosphate transport protein, ATP synthase, vacuolar-type H+-ATPase and major allergen protein significantly up-regulated in midguts, malpighian tubules, fat body of individuals which were endurable to fluoride. Our results provide useful informations for further studies on molecular mechanism of endurable to fluoride of silkworm.
2-DE and mass spectroscopy Analysis was done at the individual protein expressed difference between individuals which were sensitive to fluoride and individuals which were endurable to fluoride from the near-isogonic lines of Def gene in silkworm. 5 different protein spots that can trusted were gotten. There are 2 protein spots that are relative to energy metabolize and immunity, and we can’t confirm the function of the rest 3 protein spots. This study of this period may lay a foundation for deep researches on function of protein in future.
家蚕氟化物耐受性是很重要的一项经济性状。在蚕业生产中,家蚕是寡食性昆虫,以桑叶作为食物来源。桑叶受到氟化物的污染后造成家蚕大量死亡,对蚕桑业造成严重的危害。因此,研究家蚕的中毒机理,筛选研究家蚕耐氟基因,培育家蚕耐氟品种,并对耐氟主效基因进行定位克隆,十分重要。
本研究采用家蚕耐氟品种T6和氟化物敏感品种733新构建耐氟近等基因系。以构建好的近等基因系为材料,解剖获取回交11代的家蚕耐氟近等基因系群体中的耐氟个体和敏感个体中肠,构建家蚕耐氟近等基因系抑制消减杂交(SSH)cDNA文库。通过对抑制消减杂交cDNA文库中随机挑选的153个阳性克隆测序和聚类拼接,得到19个叠联群(contig)、48个已知功能基因(unigene)和28个未知功能基因。获得的表达序列标签(EST)经Blast比对和同源性分析,初步发现这些基因参与家蚕体内物质转运、能量代谢、基因转录、蛋白质合成与降解、蛋白质加工、信号转导、机体免疫防御、细胞结构形成等诸多生物学过程。采用实时定量RT-PCR方法,对部分基因在家蚕耐氟近等基因系群体中的耐氟个体和敏感个体的不同组织的表达进行定量分析。结果发现,磷酸根离子转运蛋白基因、ATP合成酶基因、液胞型H+-ATP酶基因以及主要过敏原蛋白基因在中肠、马氏管、脂肪体中的相对表达量有较明显的上调。实验结果可为进一步阐明家蚕耐氟的分子机制打下基础。
通过蛋白双向凝胶电泳及质谱技术,对家蚕耐氟近等基因系蛋白水平上的表达差异进行分析,最终得到了5个质谱鉴定结果可信的差异蛋白点,其中有2个蛋白点与家蚕的能量代谢和免疫防御有关,研究结果为今后展开蛋白功能方面的深入研究提供了一定基础。
China is the origin of sericulture in the world. The silk industry in our country have over 5,000 years of history, includeing many sections, such as planting mulberry, feeding silkworms, reeling silk from cocoons, knitting the silk and processing garment. Sericulture is a very important part of Chinese agriculture. Silk industry is a complete system composed of agriculture, industry and commerce. Silk exportation holds an important position in Chinese national economy.
The fluoride tolerance is the one of important economy traits in silkworm. Mulberry leaves are the major source of food for silkworm.It caused a large number of deaths to the silkworm when mulberry leaves were polluted by fluoride. This could cause serious harm to the sericulture. So it is very important to do works such as studying poisoning mechanism of fluoride in silkworm, searching for dominant endurance to fluoride(Def)gene of silkworm, breeding new new varieties of silkworm which are endurable to fluoride, map-based cloning of Def gene.
Silkworm near-isogenic lines(NILs)of dominant endurance to fluoride(Def)gene were constructed using silkworm strain 733 xin (sensitive to fluoride) and T6 (endurable to fluoride). An SSH (suppression subtractive hybridization) cDNA library was constructed between individuals which were sensitive to fluoride and individuals which were endurable to fluoride of BC11 progeny using PCR-selectTM cDNA Subtraction Kit. Plasmids were extracted from 153 positive clones randomly selected from the library and then sequenced and assembled using CAP3 software. 19contigs and 48 unigenes were obtained. Blastx searches and homology analysis with existing expressed sequence tags(ESTs)revealed that these genes were involved in many biological processes such as substance transportation, energy metabolism, gene transcription, protein synthesis or degradation, protein processing, signal transduction, organismal immunologic defence and formation of cellular construction etc. Real-time qPCR analysis confirmed that some genes, such as phosphate transport protein, ATP synthase, vacuolar-type H+-ATPase and major allergen protein significantly up-regulated in midguts, malpighian tubules, fat body of individuals which were endurable to fluoride. Our results provide useful informations for further studies on molecular mechanism of endurable to fluoride of silkworm.
2-DE and mass spectroscopy Analysis was done at the individual protein expressed difference between individuals which were sensitive to fluoride and individuals which were endurable to fluoride from the near-isogonic lines of Def gene in silkworm. 5 different protein spots that can trusted were gotten. There are 2 protein spots that are relative to energy metabolize and immunity, and we can’t confirm the function of the rest 3 protein spots. This study of this period may lay a foundation for deep researches on function of protein in future.
引文
[1]中国农业科学院蚕业研究所主编.中国养蚕学[M].上海科学技术出版社. 1990.12
[2]刘庆信,张万英.桑蚕生产中氟化物污染研究进展[J].山东农业大学学报, 1994, 25(4): 491-495.
[3]田智得.桑蚕氟化物中毒研究及防治进展综述[J].广西蚕业, 2000, 31(1): 25-30.
[4] Wang Jiaxi, Bian Yongmei. Fluoride effects on the mulberry silkworm system[J]. Envirmental Pollution, 1998, 52(1): 11-18.
[5] Chen YY. Variable tolerance of the silkworm Bombyx mori to atmospheric fluoride pollution[J]. Fluoride, 2003, 36(3): 157-162.
[6]周红亮.家蚕cyp306a1和bcl-2基因的鉴定及其耐氟性功能研究[D].江苏大学, 2008.
[7] Chen Yuyin, Sateesh kumar. Fluoride transfer in the ecosystem: fluoride loading and distribution of insects-mulberry system in a polluted site[J]. Indian Journal of Entomology, 2006, 68(3): 276-280.
[8]王志和,汤良玉,刘超.桑叶对大气氟化物吸收积累规律及其浓度预测模式探讨[J].农业环境保护, 1989, 8(1): 19-22.
[9]陈树元,卞咏梅,刘绍考.氟对农作物、家畜和蚕桑的影响[J].环境污染与防治, 1990,12(2): 10-14.
[10]曾清如,吴方正.氟污染对桑叶营养成分的影响[J].农村生态境, 1993: (3):51-53.
[11]申秀英,许晓路.氟化物对桑叶碳代谢的某些影响[J].农业环境保护, 1992:11 (6):243-248.
[12]白会钗.家蚕耐氟基因(Def)的连锁及定位分析[D].江苏科技大学, 2008.
[13] Chen YY, Du X, Jin YX. Cytochemical evidence for an anomalous dose-response of acid phosphatase activity in the blood but not the midgut of fluoride-treated silkworm larvae, Bombyx mori[J]. Fluoride, 2005, 38(2): 133-138.
[14]Miao YG, Jiang LJ, Bharathi D. Effects of fluoride on the activities of alkaline phosphatase, adenosine triphosphatase, and phosphorylase in the midgut of silkworm, Bombyx mori L [J]. 2005, 38(1): 32-37.
[15]周垂桓.受氟危害的蚕中肠及皮肤中Ca2+、Mg2+的变化[R].日蚕63次讲要. 1993, (4): 55.
[16]Chen YY. Differences in fluoride effects on fecundity among varieties of the silkworm Bombyx mori[J]. Fluoride, 2003, 36(3): 163-169.
[17]孟智启.氟化钠对家蚕中肠细胞中Ca2+浓度的影响[R].日蚕63次讲要. 1993, (4): 69.
[18]林建荣,钟生泉,史奕山.家蚕的抗氟性及其与数量性状的相关研究[J].华南农业大学学报,1992,13(3):117-120.
[19]孟智启,叶爱红,夏建国,等.浙江省桑蚕品种资源的耐氟性调查研究[J].蚕桑通报,1992, 23(4): 5-7.
[20]林健荣,钟生泉.家蚕抗氟性遗传研究Ⅱ抗氟性遗传成分分析[J].蚕业科学,1990,16(1): 25-28.
[21]张远能,刘仕贤,霍用梅,等.若干家蚕品种对六种主要蚕病的抗性鉴定[J].蚕业科学, 1982, 8(2): 94-97.
[22]王红林,郭锡杰,徐莉,等.现行蚕品种对氟化物的抗性研究[J].江苏蚕业, 1991(4): 6-9.
[23]刘庆信,牟志美,刘枷理,等.家蚕抗氟性双列杂交遗传分析[J].山东农业大学学报, 1995, 26(3): 323-326.
[24]魏兆军,王章娥,张志方.家蚕耐氟性状基因效应分析[J].蚕业科学, 2001, 27(2):158-161.
[25]林昌麒,糜懿殿,姚勤,等.家蚕耐氟显性主效基因的发现[J]. 1997, 23(4): 237-239.
[26]刘庆信,刘训理,张万英,等.家蚕抗氟性选择效果研究[J].蚕业科学,1998, 24(1): 58-59.
[27]陈克平,鲁成,向仲怀,等.家蚕耐氟性RAPD分子标记研究[J].农业生物技术学报, 2001, 9(2): 136-138.
[28]马秀康.氟化物污染对桑蚕的危害与防治对策[J].蚕桑通报, 1987, 18(2): 49-50.
[29]吴春泉,求相超,包言斐. 1986年春蚕氟化物污染情况分析[J].蚕桑通报, 1987,18 (1):9.
[30]徐安英,林昌麒,侯成香,等.家蚕耐氟春用品种华源×东升的选育及其性状[J].中国蚕业, 2006, 27(2): 30-32.
[31]林昌麒,糜懿殿,姚勤,等.家蚕耐氟遗传初探[J].江苏蚕业, 1997, 2: 7-10.
[32]徐庆刚,陈克平,姚勤等.家蚕耐氟基因RAPD分子标记的筛选及其克隆[J].生物技术, 2004, 14(3): 10-11.
[33]白会钗,徐安英,李木旺,等.利用SSR标记对家蚕耐氟基因进行连锁定位分析[J].蚕业科学, 2008, 34(2): 191-196.
[34] Heck G R, Perry S E. AG115, a MADs domain protein exoressed in developing embroyos[J]. Plan Cell, 1995(7): 1271-1282.
[35] Diatachenko L, Lau Y F C, Campbell A P,el al. Supression subtractive hybridization:A method for generating differentially regulated or tissue-specific cDNA probes and libraries.[J]. Proc Natl Sci USA, 1996(93): 6025-6030.
[36]顾克余,翟虎渠.抑制性扣除杂交技术(SSH)及其在基因克隆上的研究进展[J].生物技术通报, 1999(2): 13-16.
[37]罗文波,子淑娟,高大维.抑制性扣除杂交技术(SSH)及其研究进展[J].生物技术, 2000(10): 37-40.
[38] Clontech PCR-Select TM cDNA Subtractive Kit User Manual (PT1117-1). Published 14 February 2002 (Clontech).
[39] Van Stein O D, et al. Nucleic Acids Research [M].1997, 25(13):2598-2602
[40] George P, Yang, et al. Nucleic Acids Research [M]. 1999, 27(6):1517-1523
[41]刘春燕,王伟权,陈庆山,等.大豆花叶病毒诱导的消减文库构建及初步分析[J.生物工程学报, 2005, 21(2): 320-322.
[42] Hiroyuki T, Kazuhiro T, Gento T,et a1. Identification of genesexpressed during spore germination of Mycosphaerella pinodes[J]. JC-en Plant Pathol, 2005, 71: 190-195.
[43]曹随忠,赵兴绪,杜立新,等.抑制性消减杂交(SSH)构建乳房炎奶牛外周血白细胞差异表达cDNA文库[J].畜牧兽医学报,2005,36(6):526-530.
[44]王莹,陈葳,李旭.用抑制性消减杂交方法筛选人肾癌相关基因[J].南方医科大学学报, 2008, 28(1):89-93.
[45] Denis MG, Chadeneau C, Lecabellec MT, et a1.Overexpression of the s13 ribosomal protein in actively growing cells[J ].Int J Cancer,l993, 55:275-80.
[46] L. Anderson, J. Seilhamer, A comparison of selected mRNA and protein abundances in human liver [J]. Electrophoresis, 1997 (18):533-537
[47] Wilkins Mr, Sanchez Jc, Gooley Aa, et al. Progress with proteome projects:why all proteins expressed by a genome should be identified and how to do it[J]. Biotechnology & Genetic Engineering Reviews, 1996: 19-50.
[48]高华,高述民,孙芳芳.蛋白质组学及其在植物研究中的应用[J].安徽农学通报,2010, 16(8): 31-32.
[49] de Hoog C L,Mann M.Proteomics[J].Annu Rev Genomics Hum Genct,2004,5:267-293.
[50] Graves P R,Timothy A J.Haystead[J]. Microbiol Mol Biol Rev,2002,66(1):39-63.
[51] K J. Protein mapping by combined isoelectric focusing and electrophoresis of mouse tissue: a novel approach to testing for induced point mutations in mammals.[J]. Humangenetik, 1975(26): 231-243.
[52] O'Farrell PH. High resolusion two-dimensional electrophoresis of proteins[J]. Biol Chem, 1975, 250: 4007-4021.
[53] Govorun VM, Archakov AI. Proteomic technologies in modern biomedical science[J]. Biochemistry, 2002,10(67):1109-1123
[54] Hall R, Beale M, Fiehn O, et al. Plant metabolomics: the missing link in functional genomics strategies[J]. The Plant cell, 2002, 14(7): 1437-1440.
[55] Klose J. Protein mapping by combined isoelectric focusing and electrophoresis of mouse tissues[J]. Humangenetik, 1975, 26: 231-243
[56] Gorg A, Postel W, Gunther S. Improved horizontal two-dimensional electrophoresis with hybrid isoelectric focusing in immobilization pH gradients in the first dimension and laying-on transfer to the second dimension[J]. Electrophoresis, 1985, 6: 599-604
[57] Gorg A, Obermaier C,Boguth G, et al. The current state of two-dimensional electrophoresis with immobilized pH gradients[J]. Electrophoresis, 2000, 21(6): 1037-1053.
[58] Gorg A, Postel W, Gunther S. The current state of two-dimensional electrophoresis with immobilized PH gradients.[J]. Electrophoresis, 1988(9): 531-546.
[59] Liu J, Fang S, Li X, et al. Establishment of two-dimensional gel electrophoresis technical platform for proteome research[J]. Journal of hygiene research, 2004, 33(3):327-330.
[60] Gorg A, Weiss W, Dunn MJ. Current two-dimensional electrophoresis technology for proteomics[J]. Proteomics, 2004, 4(12): 3665-6585.
[61] Costa P, Pionneau C, Bauw G Separation and characterization of needle and xylemmaritime pine proteins[J]. Electrophoresis, 1999, 20: 1098-1108.
[62] Wijk KJV. Challenges and prospects of plant proteomics[J]. Plant Physiol, 2001 ,126(6):501-508.
[63] Peltier JB, Friso C, Kalume DE. Proteomics of chloroplast: systematic identification and targeting analysis of lumenal and peripheral thylakoid proteins[J]. Plant&cell physiology, 2000, 12(3): 319-342.
[64] Millar AH, Sweetlove L J, Giege P. Analysis of the Arobidopsis mitochondrial proteome[J]. Plant Physiol, 2001, 127(12): 1711-1727.
[65] Kruft V, Eubel H, Jansch L. Proteomic approach to identify novel mitochondrial proteins in Arobidopsis[J]. Plant Physiol, 2001, 127(12): 1694-1710.
[66] Xia Q, Zhou Z, Lu C et al. A draft sequence for the genome of the domesticated silkworm(Bombyx mori).Science, 2004, 306(5703):1937-1940.
[67] Zhong B X. Protein databank for several tissues derived fromfive instar of silkworm. Acta Genet Sin, 2001, 28 (3):217-224.
[68]侯勇,官建,赵萍等.家蚕中肠组织蛋白质组学研究[J].蚕业科学, 2007(33): 216-222.
[69]徐豫松,徐俊良,川崎秀树.家蚕脂肪体合成蛋白质变化的研究[J].蚕业科学,26(4): 239-24.
[70]颜新培,钟伯雄,徐孟奎,等.家蚕五龄后部丝腺蛋白质构成与茧层量的关系[J].蚕业科学,2003,29(4): 344-348.
[71] Schefe J H, Lehmann K E, Buschmann I R, et al. Quantitative real-time RT-PCR data analysis: current concepts and the novel“gene expression’s CT difference”formula [J]. J Mol Med, 2006, 84(11): 901-910
[72]臧荣春,吕顺霖,姚菊明,等.氟化物和VD3对家蚕幼虫中肠传输磷酸根离子的影响[J].蚕业科学,1996, 22(3): 199-201
[73]陈玉银,高其康.桑蚕幼虫中肠细胞腺苷三磷酸酶活性的细胞化学[J].浙江农业大学学报, 1996, 22(2): 157-161
[74]吕顺霖,丁春阳.氟中毒蚕体内的膜脂过氧化作用[J].浙江大学学报, 2001, 27(1): 111-112
[75]深见元弘.在氟中毒蚕的中肠内观察到微小颗粒[J].环境科学志, 1995, 8(2): 155-161
[76] Feng Q L, Ladd T R, Retnakaran A, et al. Identication and developmental expression of the mitochondrial phosphate transport protein gene from the spruce budworm, Choristoneura fumiferana[J]. Insect Biochem Mol Biol, 1998, 28(10): 791-799
[77] Hayes J D, Pulford D J. The glutathione S-transferase supergene family: regulation of GST and the contribution of the isoenzymes to cancer chemop rotection and drug resistance[J]. Crit Rev Biochem Mol Biol, 1995, 30 (6):445-600.
[78] Booth J, Borland E, Sims P. An enzyme from rat liver catalyzing conjugation with glutathione[J]. Biochem, 1961, 79: 516-524.
[79] Yamamoto K, Zhang P, Miake F et al. Cloning, expression and characterization of the theta-class glutathione S-transferase from the silkworm Bombyx mori[J]. Comp Biochem Phys part B, 2005, 141:340-346.
[80]张文吉,张友军,韩熹莱.棉铃虫不同龄期幼虫羧酸酯酶、谷胱甘肽转移酶、乙酞胆碱酯酶研究[J].植物保护学报, 1996, 23(2): 157-162.
[81]侯成香,桂仲争.家蚕谷胱甘肽硫-转移酶的组织分布及发育期变化规律[J].蚕业科学,2007, 33(3): 409-413.
[82] Matson, S W,D. W. Bean, and J. W. George. DNA helicases: enzymes with essential roles inall aspects of DNA metabolism[J]. Bioessays 1994, 16:13-22.
[83] Schmid, S, R., Linder, P. D-E-A-D protein family of putative RNA helicases[J].Mol Microbiol.1992 , 6(3): 283-91.
[84] Xu HQ, Zhang AH, Auclair C, et al. Simultaneously monitoring DNA binding and helicase-catalyzed DNA unwinding by fluorescence polarization[J]. Nucleic Acids Res, 2003, 31(14): 70.
[85] Xu HQ, Deprez E, Zhang AH, et al. The Escherichia coli RecQ helicase functions as a monomer [J]. J Biol Chem, 2003, 278 (37): 34925- 34933.
[86] Shiratori A,Shibata M,Arisawa F, Hanaoka Y,Murakami T,Eki. Systematic identification, classification, and characterization of the open reading frames which encode novel helicase-related proteins in Saccharomyces cerevisiae by gene disruption and Northern analysis[J].Yeast ,1999 ,15:219-253.
[2]刘庆信,张万英.桑蚕生产中氟化物污染研究进展[J].山东农业大学学报, 1994, 25(4): 491-495.
[3]田智得.桑蚕氟化物中毒研究及防治进展综述[J].广西蚕业, 2000, 31(1): 25-30.
[4] Wang Jiaxi, Bian Yongmei. Fluoride effects on the mulberry silkworm system[J]. Envirmental Pollution, 1998, 52(1): 11-18.
[5] Chen YY. Variable tolerance of the silkworm Bombyx mori to atmospheric fluoride pollution[J]. Fluoride, 2003, 36(3): 157-162.
[6]周红亮.家蚕cyp306a1和bcl-2基因的鉴定及其耐氟性功能研究[D].江苏大学, 2008.
[7] Chen Yuyin, Sateesh kumar. Fluoride transfer in the ecosystem: fluoride loading and distribution of insects-mulberry system in a polluted site[J]. Indian Journal of Entomology, 2006, 68(3): 276-280.
[8]王志和,汤良玉,刘超.桑叶对大气氟化物吸收积累规律及其浓度预测模式探讨[J].农业环境保护, 1989, 8(1): 19-22.
[9]陈树元,卞咏梅,刘绍考.氟对农作物、家畜和蚕桑的影响[J].环境污染与防治, 1990,12(2): 10-14.
[10]曾清如,吴方正.氟污染对桑叶营养成分的影响[J].农村生态境, 1993: (3):51-53.
[11]申秀英,许晓路.氟化物对桑叶碳代谢的某些影响[J].农业环境保护, 1992:11 (6):243-248.
[12]白会钗.家蚕耐氟基因(Def)的连锁及定位分析[D].江苏科技大学, 2008.
[13] Chen YY, Du X, Jin YX. Cytochemical evidence for an anomalous dose-response of acid phosphatase activity in the blood but not the midgut of fluoride-treated silkworm larvae, Bombyx mori[J]. Fluoride, 2005, 38(2): 133-138.
[14]Miao YG, Jiang LJ, Bharathi D. Effects of fluoride on the activities of alkaline phosphatase, adenosine triphosphatase, and phosphorylase in the midgut of silkworm, Bombyx mori L [J]. 2005, 38(1): 32-37.
[15]周垂桓.受氟危害的蚕中肠及皮肤中Ca2+、Mg2+的变化[R].日蚕63次讲要. 1993, (4): 55.
[16]Chen YY. Differences in fluoride effects on fecundity among varieties of the silkworm Bombyx mori[J]. Fluoride, 2003, 36(3): 163-169.
[17]孟智启.氟化钠对家蚕中肠细胞中Ca2+浓度的影响[R].日蚕63次讲要. 1993, (4): 69.
[18]林建荣,钟生泉,史奕山.家蚕的抗氟性及其与数量性状的相关研究[J].华南农业大学学报,1992,13(3):117-120.
[19]孟智启,叶爱红,夏建国,等.浙江省桑蚕品种资源的耐氟性调查研究[J].蚕桑通报,1992, 23(4): 5-7.
[20]林健荣,钟生泉.家蚕抗氟性遗传研究Ⅱ抗氟性遗传成分分析[J].蚕业科学,1990,16(1): 25-28.
[21]张远能,刘仕贤,霍用梅,等.若干家蚕品种对六种主要蚕病的抗性鉴定[J].蚕业科学, 1982, 8(2): 94-97.
[22]王红林,郭锡杰,徐莉,等.现行蚕品种对氟化物的抗性研究[J].江苏蚕业, 1991(4): 6-9.
[23]刘庆信,牟志美,刘枷理,等.家蚕抗氟性双列杂交遗传分析[J].山东农业大学学报, 1995, 26(3): 323-326.
[24]魏兆军,王章娥,张志方.家蚕耐氟性状基因效应分析[J].蚕业科学, 2001, 27(2):158-161.
[25]林昌麒,糜懿殿,姚勤,等.家蚕耐氟显性主效基因的发现[J]. 1997, 23(4): 237-239.
[26]刘庆信,刘训理,张万英,等.家蚕抗氟性选择效果研究[J].蚕业科学,1998, 24(1): 58-59.
[27]陈克平,鲁成,向仲怀,等.家蚕耐氟性RAPD分子标记研究[J].农业生物技术学报, 2001, 9(2): 136-138.
[28]马秀康.氟化物污染对桑蚕的危害与防治对策[J].蚕桑通报, 1987, 18(2): 49-50.
[29]吴春泉,求相超,包言斐. 1986年春蚕氟化物污染情况分析[J].蚕桑通报, 1987,18 (1):9.
[30]徐安英,林昌麒,侯成香,等.家蚕耐氟春用品种华源×东升的选育及其性状[J].中国蚕业, 2006, 27(2): 30-32.
[31]林昌麒,糜懿殿,姚勤,等.家蚕耐氟遗传初探[J].江苏蚕业, 1997, 2: 7-10.
[32]徐庆刚,陈克平,姚勤等.家蚕耐氟基因RAPD分子标记的筛选及其克隆[J].生物技术, 2004, 14(3): 10-11.
[33]白会钗,徐安英,李木旺,等.利用SSR标记对家蚕耐氟基因进行连锁定位分析[J].蚕业科学, 2008, 34(2): 191-196.
[34] Heck G R, Perry S E. AG115, a MADs domain protein exoressed in developing embroyos[J]. Plan Cell, 1995(7): 1271-1282.
[35] Diatachenko L, Lau Y F C, Campbell A P,el al. Supression subtractive hybridization:A method for generating differentially regulated or tissue-specific cDNA probes and libraries.[J]. Proc Natl Sci USA, 1996(93): 6025-6030.
[36]顾克余,翟虎渠.抑制性扣除杂交技术(SSH)及其在基因克隆上的研究进展[J].生物技术通报, 1999(2): 13-16.
[37]罗文波,子淑娟,高大维.抑制性扣除杂交技术(SSH)及其研究进展[J].生物技术, 2000(10): 37-40.
[38] Clontech PCR-Select TM cDNA Subtractive Kit User Manual (PT1117-1). Published 14 February 2002 (Clontech).
[39] Van Stein O D, et al. Nucleic Acids Research [M].1997, 25(13):2598-2602
[40] George P, Yang, et al. Nucleic Acids Research [M]. 1999, 27(6):1517-1523
[41]刘春燕,王伟权,陈庆山,等.大豆花叶病毒诱导的消减文库构建及初步分析[J.生物工程学报, 2005, 21(2): 320-322.
[42] Hiroyuki T, Kazuhiro T, Gento T,et a1. Identification of genesexpressed during spore germination of Mycosphaerella pinodes[J]. JC-en Plant Pathol, 2005, 71: 190-195.
[43]曹随忠,赵兴绪,杜立新,等.抑制性消减杂交(SSH)构建乳房炎奶牛外周血白细胞差异表达cDNA文库[J].畜牧兽医学报,2005,36(6):526-530.
[44]王莹,陈葳,李旭.用抑制性消减杂交方法筛选人肾癌相关基因[J].南方医科大学学报, 2008, 28(1):89-93.
[45] Denis MG, Chadeneau C, Lecabellec MT, et a1.Overexpression of the s13 ribosomal protein in actively growing cells[J ].Int J Cancer,l993, 55:275-80.
[46] L. Anderson, J. Seilhamer, A comparison of selected mRNA and protein abundances in human liver [J]. Electrophoresis, 1997 (18):533-537
[47] Wilkins Mr, Sanchez Jc, Gooley Aa, et al. Progress with proteome projects:why all proteins expressed by a genome should be identified and how to do it[J]. Biotechnology & Genetic Engineering Reviews, 1996: 19-50.
[48]高华,高述民,孙芳芳.蛋白质组学及其在植物研究中的应用[J].安徽农学通报,2010, 16(8): 31-32.
[49] de Hoog C L,Mann M.Proteomics[J].Annu Rev Genomics Hum Genct,2004,5:267-293.
[50] Graves P R,Timothy A J.Haystead[J]. Microbiol Mol Biol Rev,2002,66(1):39-63.
[51] K J. Protein mapping by combined isoelectric focusing and electrophoresis of mouse tissue: a novel approach to testing for induced point mutations in mammals.[J]. Humangenetik, 1975(26): 231-243.
[52] O'Farrell PH. High resolusion two-dimensional electrophoresis of proteins[J]. Biol Chem, 1975, 250: 4007-4021.
[53] Govorun VM, Archakov AI. Proteomic technologies in modern biomedical science[J]. Biochemistry, 2002,10(67):1109-1123
[54] Hall R, Beale M, Fiehn O, et al. Plant metabolomics: the missing link in functional genomics strategies[J]. The Plant cell, 2002, 14(7): 1437-1440.
[55] Klose J. Protein mapping by combined isoelectric focusing and electrophoresis of mouse tissues[J]. Humangenetik, 1975, 26: 231-243
[56] Gorg A, Postel W, Gunther S. Improved horizontal two-dimensional electrophoresis with hybrid isoelectric focusing in immobilization pH gradients in the first dimension and laying-on transfer to the second dimension[J]. Electrophoresis, 1985, 6: 599-604
[57] Gorg A, Obermaier C,Boguth G, et al. The current state of two-dimensional electrophoresis with immobilized pH gradients[J]. Electrophoresis, 2000, 21(6): 1037-1053.
[58] Gorg A, Postel W, Gunther S. The current state of two-dimensional electrophoresis with immobilized PH gradients.[J]. Electrophoresis, 1988(9): 531-546.
[59] Liu J, Fang S, Li X, et al. Establishment of two-dimensional gel electrophoresis technical platform for proteome research[J]. Journal of hygiene research, 2004, 33(3):327-330.
[60] Gorg A, Weiss W, Dunn MJ. Current two-dimensional electrophoresis technology for proteomics[J]. Proteomics, 2004, 4(12): 3665-6585.
[61] Costa P, Pionneau C, Bauw G Separation and characterization of needle and xylemmaritime pine proteins[J]. Electrophoresis, 1999, 20: 1098-1108.
[62] Wijk KJV. Challenges and prospects of plant proteomics[J]. Plant Physiol, 2001 ,126(6):501-508.
[63] Peltier JB, Friso C, Kalume DE. Proteomics of chloroplast: systematic identification and targeting analysis of lumenal and peripheral thylakoid proteins[J]. Plant&cell physiology, 2000, 12(3): 319-342.
[64] Millar AH, Sweetlove L J, Giege P. Analysis of the Arobidopsis mitochondrial proteome[J]. Plant Physiol, 2001, 127(12): 1711-1727.
[65] Kruft V, Eubel H, Jansch L. Proteomic approach to identify novel mitochondrial proteins in Arobidopsis[J]. Plant Physiol, 2001, 127(12): 1694-1710.
[66] Xia Q, Zhou Z, Lu C et al. A draft sequence for the genome of the domesticated silkworm(Bombyx mori).Science, 2004, 306(5703):1937-1940.
[67] Zhong B X. Protein databank for several tissues derived fromfive instar of silkworm. Acta Genet Sin, 2001, 28 (3):217-224.
[68]侯勇,官建,赵萍等.家蚕中肠组织蛋白质组学研究[J].蚕业科学, 2007(33): 216-222.
[69]徐豫松,徐俊良,川崎秀树.家蚕脂肪体合成蛋白质变化的研究[J].蚕业科学,26(4): 239-24.
[70]颜新培,钟伯雄,徐孟奎,等.家蚕五龄后部丝腺蛋白质构成与茧层量的关系[J].蚕业科学,2003,29(4): 344-348.
[71] Schefe J H, Lehmann K E, Buschmann I R, et al. Quantitative real-time RT-PCR data analysis: current concepts and the novel“gene expression’s CT difference”formula [J]. J Mol Med, 2006, 84(11): 901-910
[72]臧荣春,吕顺霖,姚菊明,等.氟化物和VD3对家蚕幼虫中肠传输磷酸根离子的影响[J].蚕业科学,1996, 22(3): 199-201
[73]陈玉银,高其康.桑蚕幼虫中肠细胞腺苷三磷酸酶活性的细胞化学[J].浙江农业大学学报, 1996, 22(2): 157-161
[74]吕顺霖,丁春阳.氟中毒蚕体内的膜脂过氧化作用[J].浙江大学学报, 2001, 27(1): 111-112
[75]深见元弘.在氟中毒蚕的中肠内观察到微小颗粒[J].环境科学志, 1995, 8(2): 155-161
[76] Feng Q L, Ladd T R, Retnakaran A, et al. Identication and developmental expression of the mitochondrial phosphate transport protein gene from the spruce budworm, Choristoneura fumiferana[J]. Insect Biochem Mol Biol, 1998, 28(10): 791-799
[77] Hayes J D, Pulford D J. The glutathione S-transferase supergene family: regulation of GST and the contribution of the isoenzymes to cancer chemop rotection and drug resistance[J]. Crit Rev Biochem Mol Biol, 1995, 30 (6):445-600.
[78] Booth J, Borland E, Sims P. An enzyme from rat liver catalyzing conjugation with glutathione[J]. Biochem, 1961, 79: 516-524.
[79] Yamamoto K, Zhang P, Miake F et al. Cloning, expression and characterization of the theta-class glutathione S-transferase from the silkworm Bombyx mori[J]. Comp Biochem Phys part B, 2005, 141:340-346.
[80]张文吉,张友军,韩熹莱.棉铃虫不同龄期幼虫羧酸酯酶、谷胱甘肽转移酶、乙酞胆碱酯酶研究[J].植物保护学报, 1996, 23(2): 157-162.
[81]侯成香,桂仲争.家蚕谷胱甘肽硫-转移酶的组织分布及发育期变化规律[J].蚕业科学,2007, 33(3): 409-413.
[82] Matson, S W,D. W. Bean, and J. W. George. DNA helicases: enzymes with essential roles inall aspects of DNA metabolism[J]. Bioessays 1994, 16:13-22.
[83] Schmid, S, R., Linder, P. D-E-A-D protein family of putative RNA helicases[J].Mol Microbiol.1992 , 6(3): 283-91.
[84] Xu HQ, Zhang AH, Auclair C, et al. Simultaneously monitoring DNA binding and helicase-catalyzed DNA unwinding by fluorescence polarization[J]. Nucleic Acids Res, 2003, 31(14): 70.
[85] Xu HQ, Deprez E, Zhang AH, et al. The Escherichia coli RecQ helicase functions as a monomer [J]. J Biol Chem, 2003, 278 (37): 34925- 34933.
[86] Shiratori A,Shibata M,Arisawa F, Hanaoka Y,Murakami T,Eki. Systematic identification, classification, and characterization of the open reading frames which encode novel helicase-related proteins in Saccharomyces cerevisiae by gene disruption and Northern analysis[J].Yeast ,1999 ,15:219-253.